Project Summary This proposal will focus on the development of tolerogenic vaccines that elicit responses of FOXP3+ regulatory T cells (Tregs) in murine models of EAE. This program will develop novel tolerogenic vaccines based on the use of Interferon- beta (IFN-?) as a tolerogenic adjuvant. Preliminary data indicated that these tolerogenic vaccines elicited a major FOXP3+ regulatory T cell (Treg) population because a single tolerogenic vaccination directly elicited FOXP3+ Tregs in vivo. Treatment of mice with an anti-CD25 mAb that depleted Tregs reversed vaccine-induced tolerance. Also, IFN-?- induced Tregs mediated the adoptive transfer of resistance to EAE. Three repeated immunizations with the tolerogenic vaccine elicited a persistent memory/ effector Treg subset. Our hypothesis is that tolerogenic vaccination drives tolerance via the induction of FOXP3+ Tregs and culminates in the induction of CTLA-4+ effector Tregs and a mechanism of infec- tious tolerance. Aim 1 will test whether tolerogenic vaccination elicits polyclonal tolerance to NAg peptides by a mecha- nism dependent upon NAg-specific FOXP3+ Tregs. The approach will test multiple myelin peptides for tolerogenic vac- cination of transgenic TCR model systems in combination with FOXP3-reporter, fate-mapping Treg models and Treg de- pletion strategies to assess tolerance induction, function, phenotype, and stability of vaccine-induced Treg populations. Aim 2 will assess induction and effector mechanisms of IFN-? induced FOXP3+ Tregs. Aim 2a will test whether IFN-? based tolerogenic vaccines elicit `nave', `central' or `effector' FOXP3+ T cells when the Type I IFNAR is absent in mye- loid cells, dendritic cells, pan T cells, or FOXP3+ T cells. Aim 2b will test whether IFN-? and NAg elicit FOXP3+ T cells that express the hallmarks infectious tolerance. The aim is based on the observation that CD45.2 FOXP3+ 2D2-FIG Tregs effectively directed the nascent differentiation of a nave population of FOXP3(null) CD45.1 T cells into a major FOXP3+ population in mixed activation cultures. The proposed research will provide novel information on tolerogenic vaccination and infectious tolerance via suppressive epitope spread among the major, MS-associated myelin NAg epitopes.